Immunohistochemistry with Fluorescence Detection

Immunohistochemistry with Fluorescence detection

This protocol describes standard immunohistochemistry (IHC) of (para)formaldehyde-fixed paraffin-embedded tissue sections using optional autoclaving for antigen retrieval and fluorescence detection.

1. Tissue fixation, embedding, and sectioning

● Remove piece of tissue using forceps, scissors, scalpel. Briefly rinse or wash with 0.9% NaCl and transfer to fixation solution. ● For IHC fix tissue in 4% PFA in TBS at 4°C for 2-24 h. Fixation time depends on the size of the sample and should be long enough for complete penetration of the sample by the fixation solution. Over-fixation should be avoided because this leads to masking of the antigens and poor reactivity. ● Use automatic dehydration and embed the sample in paraffin. Paraffin blocks can be stored at room temperature. ● Using a microtome cut sections of 4-6 µm and transfer sections to slides with adherent coating. Label slides and dry slides for 2-16 h at 50°C in an incubator.

2. Immunohistochemical staining

● Remove paraffin by incubating slides for 3 min each in: 4x Xylol - 100% EtOH - 96% EtOH - 80% EtOH. ● Transfer slides to a Tray and rinse for 5 min in running tap water. ● Transfer slides to a black PP Tray filled with 10mM Na-citrate . Heat in an autoclave for 10 min at 121°C. ● When the slides have cooled so you can hold them transfer slides to a tray filled with TBS . Wash slides twice in TBS for 5 min. ● Remove slides from tray and mount them on Coverplates for subsequent incubations. Wash once with TBS . ● For blocking endogenous peroxidase activity add 150 µl 1% H 2O2 and incubate 15 min at RT. ● Wash 3 x with TNT . ● For blocking non-specific sites add 150 µl TNB and incubate 30 min at RT. ● Add 150 µl of 1st Antibody , incubate o/n at 4°C (or for a suitable time at RT). ● Wash 3 x with TNT . ● Carry out subsequent steps in the dark! ● Add 150 µl of 2nd Antibody-FITC (or other fluorescence label), incubate 60-120 min at RT. (Alternatively: add 150 µl 2 nd Antibody-Biotin - incubate 30-60 min at RT - wash 3 x with TNT - add 150 µl Streptavidin-FITC - incubate 30-60 min at RT) ● Wash 3 x with TNT . ● Disassemble slides from Coverplates, carefully remove liquid. ● Add 100 µl PI (or DAPI) (1µg/ml) to completely cover the section and incubate 5 min at RT. ● Remove PI (DAPI) (toxic waste!) and incubate slides 5 min in a Tray containing TBS. ● Mount coverslips using an aqueous-based Fluorescence Mounting Medium and store slides in the dark.

3. Materials and reagents

Glass Trays and PP Trays for slides and suitable slide holders Coverplates and Coverplate Racks (Shandon, plastic incubation holders for slides, incubation volume ca. 100 µl) 4% PFA in TBS : dissolve 20g Paraformaldehyde (Merck) in 500ml TBS by heating and stirring for several hours, filter and store at 4°C Xylol , 100% Ethanol , 96% Ethanol , 80% Ethanol , 70% Ethanol (these can be reused for several weeks, use separate series of solutions for paraffin removal and dehydration) 200mM Na-citrate : 200mM Citric acid monohydrate (42g/l), pH=6.0 (NaOH)

10mM Na-citrate : 50ml 200mM Na-citrate + 950ml H 2O 10xTBS : 500mM Tris (60.55g/l), 1500mM NaCl (87.66g/l), pH=7.5 (HCl)

TBS : 100ml 10xTBS + 900ml H 2O

1% H 2O2: 33µl 30% H 2O2 (Merck) + 967µl TBS (prepare fresh, store at 4°C not longer than a week) TNT : TBS + 0.05% Tween 20 (500µl/l) TNB : TBS + 0.5% Blocking Reagent (500mg/100ml, from TSA-Kit (NEN)) dissolve by continuous stirring while heating up to 60°C, store aliquots at -20°C 1st Antibody : specific for antigen to be detected, diluted appropriately in TNB before use

2nd Antibody-FITC : FITC-conjugate (or other label) specific for 1 st Ab, diluted appropriately in TNB before use 2nd Antibody-Biotin : Biotin-conjugate specific for 1 st Ab, diluted appropriately in TNB before use Streptavidin-FITC : Streptavidin-FITC-conjugate, diluted appropriately in TNB before use

PI : Propidium iodide, Stock = 50µg/ml H 2O, Working solution = 1µg/ml H 2O DNA stain that produces red fluorescence with green illumination (Ex/Em=535/617nm)

DAPI : 4',6-Diamidino-2-phenylindole, Stock = 50µg/ml H 2O, Working solution = 1µg/ml H 2O DNA stain that produces blue fluorescence with UV illumination (Ex/Em=358/461nm) Mounting Medium : Fluorescence Mounting Medium (Dako S3023), reduces fading of fluorescence

Fluorescence labels : FITC = Fluorescein isothiocyanate (Ex/Em=495/525nm) (blue > green) TRITC = Tetramethyl rhodamine isothiocyanate (Ex/Em=552/570nm) (green > red-orange) RPE = R-phycoerythrin (Ex/Em=480-545-565/620nm) (green > orange-red)

CONTRIBUTED BY: Hubert Schwelberger ([email protected]) LAST MODIFIED: 2012-06-20